Dynamic transcriptional and epigenetic changes drive cellular plasticity in the liver

Background and aims Following liver injury, a fraction of hepatocytes adopt features of biliary epithelial cells (BECs) in a process known as biliary reprogramming. The aim of this study was to elucidate the molecular events accompanying this dramatic shift in cellular identity. Approach and Results We applied the techniques of bulk RNA-sequencing (RNA-seq), single cell RNA-sequencing (scRNA-seq), and assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) to define the epigenetic and transcriptional changes associated with biliary reprogramming. In addition, we examined the role of TGFβ signaling by profiling cells undergoing reprogramming in mice with hepatocyte-specific deletion in the downstream TGFβ signaling component Smad4. Biliary reprogramming followed a stereotyped pattern of altered gene expression consisting of robust induction of biliary genes and weaker repression of hepatocyte genes. These changes in gene expression were accompanied by corresponding modifications at the chromatin level. While some reprogrammed cells had molecular features of "fully differentiated" BECs, most lacked some biliary characteristics and retained some hepatocyte characteristics. Surprisingly, single cell analysis of Smad4 mutant mice revealed a dramatic increase in reprogramming. Conclusion Hepatocytes undergo widespread chromatin and transcriptional changes during biliary reprogramming, resulting in epigenetic and gene expression profiles that are similar to, but distinct from, native BECs. Reprogramming involves a progressive accumulation of biliary molecular features without discrete intermediates. Paradoxically, canonical TGFβ signaling through Smad4 appears to constrain biliary reprogramming, indicating that TGFβ can either promote or inhibit biliary differentiation depending on which downstream components of the pathway are engaged. This work has implications for the formation of new BECs and bile ducts in the adult liver.